Clones of mutant cells marked with green fluorescent protein (GFP) can be generated in the eye-antenna imaginal discs of Drosophila larvae by mitotic recombination. Clones expressing the oncogenic protein Ras^V12 moderately overgrow ¹² (Fig. 1a, b). Clones mutant for scrib lose apico-basal polarity and die ^6,13 (Fig. 1c). In contrast, scrib clones simultaneously expressing Ras^V12 grow into large metastatic tumours (Fig. 1d) ⁸. To better understand cooperation between these two mutations, we produced animals in which cell division after a mitotic recombination event creates two daughter cells, one expressing Ras^V12 and the other mutant for scrib. Discs containing adjacent Ras^V12 (GFP-positive) and scrib⁻ clones developed into large tumours, capable of invading the ventral nerve cord (VNC) (Fig. 1e). This shows that Ras^V12 and scrib cooperate for tumour induction also when they occur in different cells. We will refer to these tumours as Ras^V12//scrib⁻ tumours, to denote interclonal oncogenic cooperation and distinguish them from Ras^V12scrib⁻ tumours, in which cooperation occurs in the same cells intraclonally.

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Figure 1

Interclonal cooperation between Ras^V12 and scrib⁻ causes tumours

a-e, Clones of cells marked with GFP in the eye-antennal discs of third-instar larvae. Upper subpanels show the cephalic complex (CC), consisting of eye-antennal discs (EA), brain (B) and ventral nerve cord (VNC). Lower subpanels show the dissected VNC. Compared to wild-type clones (a), Ras^V12 clones overgrow moderately (b). scrib⁻ clones are eliminated from the tissue (c). Double mutant Ras^V12scrib⁻ clones (d, intraclonal cooperation), as well as Ras^V12 clones confronted with scrib⁻ clones (e, interclonal cooperation), cause tumours that overgrow and invade the VNC. f-i, Confocal sections of the inner tumour mass in Ras^V12scrib⁻ (f, g) and Ras^V12//scrib⁻ tumours (h, i) at day 6 and 14 after egg laying (AEL). Non-Ras^V12 cells (absence of GFP) are progressively eliminated from the tissue. Cell nuclei labeled with DAPI. Yellow arrowheads point to scattered remaining GFP-negative cells (insets in g and i).

In the late stages of the development of Ras^V12//scrib⁻ tumours, most cells in the tumour mass are Ras^V12 cells (Fig. 1h, i). scrib⁻ cells, as well as residual wild-type cells, are almost completely absent from the tissue, similar to the absence of wild-type cells in late Ras^V12scrib⁻ tumours (Fig. 1f, g). To test the possibility that Ras^V12//scrib⁻ tumours are caused by unopposed growth of Ras^V12 cells, we examined eye-antennal discs where all cells expressed Ras^V12. Dramatic overgrowth or invasion did not occur (Supplementary Fig. 1), showing that interaction between Ras^V12 and scrib⁻ cells is required for tumour development. Interclonal cooperation between Ras^V12 and lethal giant larvae (lgl) also produced tumours (Supplementary Fig. 2), suggesting that other polarity mutations can cooperate interclonally with Ras^V12. Intrigued by these findings, we decided to investigate the mechanisms underlying non-autonomous oncogenic cooperation and sustained growth in Ras^V12//scrib⁻ tumours.

JAK/STAT signaling promotes cell proliferation in different contexts in mammals and flies ¹⁴, including the overgrowth caused by mutation of several tumour suppressors ¹⁵. In a cDNA microarray analysis of Ras^V12scrib⁻ tumours, we discovered upregulation of the unpaired genes (upd, upd2 and upd3; data not shown), which encode JAK/STAT-activating cytokines related to Interleukin 6 ^16-18. We confirmed the upregulation of the unpaired genes in Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours by real-time RT-PCR (Fig. 2a). Furthermore, we observed elevated expression of the JAK/STAT reporter STAT-GFP ¹⁹ in both Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours (Fig. 2b-e), thus correlating high expression of Upd cytokines with increased JAK/STAT activity.

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Figure 2

Synergy between Ras and JAK/STAT signaling promotes growth and invasion in Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours

a, Quantification by real-time RT-PCR of expression of the upd genes, encoding the JAK/STAT-activating cytokines Upd, Upd2 and Upd3, in eye-antennal discs containing wild-type clones, Ras^V12-expressing clones, scrib⁻ clones (day 6 AEL), Ras^V12scrib− tumours and Ras^V12//scrib⁻ tumours (day 10 AEL). Expression is normalized to the housekeeping gene rp49. Error bars depict 95% confidence intervals (1.96 × s.e.m., n=3). b-e, Expression of the JAK/STAT reporter STAT-GFP (green) in day 6 AEL eye-antennal discs containing wild-type clones (b, red), Ras^V12 clones (c), Ras^V12scrib⁻ (d) and Ras^V12//scrib⁻ tumours (e). f, g, Suppression of Ras^V12scrib⁻ (f) and Ras^V12//scrib⁻ (g) tumours by expression of a dominant negative form of the JAK/STAT receptor Domeless (Dome^DN). h, Clones overexpressing Upd. i-l, Tumours caused by Ras^V12 clones co-overexpressing Upd (i), Upd2 (j), Upd3 (k) and both Upd and Upd2 (l). m, Ras^V12Upd tumours are suppressed by Dome^DN.

To test the involvement of JAK/STAT signaling in the growth of Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours, we used a dominant negative form of the JAK/STAT receptor Domeless (Dome^DN) ²⁰. Expression of Dome^DN achieved suppression of overgrowth and invasion of the VNC in Ras^V12scrib⁻ tumours (Fig. 2f). Also Ras^V12//scrib⁻ tumours were suppressed by expression of Dome^DN in Ras^V12 cells (Fig. 2g). A loss-of-function mutation in stat92E, encoding the Drosophila STAT transcriptional activator, reduced growth and invasiveness of Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours (Supplementary Fig. 3). From these experiments, we conclude that JAK/STAT signaling is required for the development of Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours.

The suppression of Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours by reducing JAK/STAT activity in Ras^V12 cells points to cooperation between Ras and JAK/STAT signaling as a cause of tumour growth. To confirm this, we generated clones of cells co-expressing Upd cytokines and Ras^V12. While Upd overexpression in wild-type cells (Fig. 2h), in scrib⁻ cells or in wild-type cells adjacent to scrib⁻ cells (Supplementary Fig. 4) did not cause tumours, co-expression of Ras^V12 and Upd produced large invasive tumours (Fig. 2i). Similar results were obtained co-expressing Ras^V12 and Upd2 (Fig. 2j), whereas co-expression of Ras^V12 and Upd3 caused smaller, non-invasive tumours (Fig. 2k). Finally, Ras^V12Upd Upd2 tumours were larger than Ras^V12Upd and Ras^V12Upd2 tumours (Fig. 2l), suggesting an additive effect of the expression of different Upd cytokines (see also Supplementary Fig. 5).

Prevention of actual cell death in cells apoptotically stimulated has been shown to potently promote overgrowth ²¹. To assess a possible involvement of apoptosis prevention in the synergy between Ras and JAK/STAT signaling, we coexpressed the apoptosis inhibitor p35 with Ras^V12 or Upd. Neither conditions produced tumours (Supplementary Fig. 6), suggesting that cooperation between Ras and JAK/STAT involves a mechanism other than apoptosis prevention. Ras^V12Upd tumours were suppressed by expression of Dome^DN (Fig. 2m), thus confirming that their development requires JAK/STAT activity. In all, both loss- and gain-of-function experiments lead us to conclude that Ras and JAK/STAT signaling exhibit a strong synergistic tumour-promoting interaction, responsible for the development of Ras^V12scrib⁻ and Ras^V12//scrib− tumours.

Having established the involvement of JAK/STAT signaling in the growth of Ras^V12scrib− and Ras^V12//scrib⁻ tumours, we decided to investigate how expression of the Upd cytokines is upregulated. We previously showed that expression of the unpaired genes is elevated in wounds in a JNK-dependent manner ²². It has been shown as well that JNK signaling can induce non-autonomous overgrowth ^23,24 and that JNK signaling is upregulated in scrib⁻ clones ^13,25 and scrib⁻ discs ²², which develop as tumours in scrib⁻ larvae ⁶. To test the possibility that JNK activation causes ectopic JAK/STAT signaling in scrib⁻ cells, we monitored STAT-GFP expression in discs double mutant for scrib and hep, coding for the Drosophila JNK-kinase Hemipterous. In these discs, STAT-GFP expression was reduced and overgrowth suppressed (Fig. 3a-c), showing that JAK/STAT elevation in scrib⁻ cells depends on JNK activity.

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Figure 3

JNK signaling drives oncogenic cooperation upstream of JAK/STAT

a-c, STAT-GFP expression in wing discs of wild-type larvae (a) and scrib⁻ larvae heterozygous (b) or hemizygous (male, c) for the JNK-kinase loss-of-function mutation hep^r75. Overgrowth and STAT-GFP upregulation are suppressed by hep^r75. Arrowheads point to normal STAT-GFP expression in the wing hinge. Discs stained with phalloidin (red). d-g, Expression of a dominant negative form of the Jun-kinase Basket (Bsk^DN) suppresses Ras^V12scrib⁻ tumours (d, e), but not Ras^V12Upd tumours (f, g). h, i, Expression of Bsk^DN in Ras^V12 cells partially suppresses Ras^V12//scrib⁻ tumours. j, Quantification by real-time RT-PCR of expression of the upd genes in Ras^V12//scrib⁻ and Ras^V12Bsk^DN//scrib⁻ tumours (day 6 AEL). Error bars represent 95% confidence intervals (n=3). k, Propagation of JNK activity (puc-lacZ reporter, green) into the posterior (P) compartment (arrowhead) in wing discs wounded in the anterior (A) compartment (asterisk) 24 hours after wounding. l, puc-lacZ expression in discs expressing the JNK-phosphatase Puc under control of ptc-GAL4 (red cells, expressing RFP), wounded as in k. Puc is both a downstream target and a negative regulator of JNK. Propagation of puc-lacZ expression into the P compartment is not observed (hollow arrowhead).

The induction of Upd cytokines by JNK in scrib⁻ cells can explain the growth of Ras^V12scrib⁻ tumours, placing JAK-STAT signaling downstream of JNK. In support of this, a dominant negative form of the Jun-kinase Basket (Bsk^DN) suppressed Ras^V12scrib− tumours ⁹ (Fig. 3d, e), but not Ras^V12Upd tumours (Fig. 3f, g). In the case of Ras^V12//scrib⁻ tumours, few scrib⁻ cells remain in the tissue at late stages (Fig. 1i). Therefore, Upd induction in scrib⁻ cells cannot fully account for tumour development. Indeed, expression of Bsk^DN in Ras^V12 cells partially suppressed the growth of Ras^V12//scrib⁻ tumours (Fig. 3h, i) and expression of the unpaired genes (Fig. 3j). This shows that in Ras^V12//scrib⁻ tumours expression of Upd cytokines downstream of JNK signaling also occurs in Ras^V12 cells.

scrib⁻ clones cause JNK activation both autonomously and non-autonomously ²⁵. Furthermore, in wing discs, wounding induces JNK activation away from the site of wounding ^22,26, suggesting that JNK activity can propagate. To investigate this, we wounded wing discs and examined the expression of puckered (puc), a JNK downstream gene encoding a JNK-phosphatase that negatively regulates the pathway ²⁷. When wounds were induced in the anterior or posterior wing regions, JNK activation, revealed by puc-lacZ expression, was observed across the disc in the opposite compartment (Fig. 3k). In contrast, overexpression of Puc in a central stripe of cells prevented expansion of JNK to the opposite compartment. (Fig. 3l, Supplemental Fig. 7). This indicates that JNK activity propagates through a feed-forward loop and, together with previous findings, suggests that in Ras^V12//scrib⁻ tumours, scrib⁻ cells trigger JNK activation and that this activation propagates to adjacent Ras^V12 cells. JNK-dependent upregulation of Upd cytokines in Ras^V12 cells, thus, can sustain tumour growth when the original source of JNK activity, the scrib⁻ cells, is no longer present.

The previous experiments reveal a central role for JNK in the cooperation of Ras^V12 and scrib⁻. Since both wounds and scrib⁻ induce JNK activation, we tested the possibility that tissue damage could cooperate with Ras^V12 to promote tumour overgrowth. We wounded larval right wing discs and examined them 48 hours later. In wild-type discs, compared to the unwounded left disc, wounding resulted in size reduction (Fig 4a, b; Supplementary Fig. 8; wounded/unwounded size ratio ±SD=0.70±0.18). In contrast, wounding of Ras^V12-expressing discs caused a marked increase in Ras^V12-induced overgrowth (Fig. 4c, d; Supplementary Fig. 8; 1.46±0.31). No metastasis was detected in this experiment (not shown). Finally, the wounded/unwounded ratio in p35-expressing discs (1.09±0.14, Supplementary Fig. 8) shows that apoptosis prevention by Ras^V12 cannot completely account for its cooperation with mechanically-induced damage.

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Figure 4

Tissue damage, compensatory growth and a model for interclonal oncogenic cooperation

a-d, Cooperation between Ras^V12 and tissue damage. Right (wounded) and left (unwounded) wing discs of a wild-type larva (a, b) and a larva expressing Ras^V12 under control of nub-GAL4 (c, d). Discs were wounded by repeated pinching and dissected 48h later. Expression of RFP driven by nub-GAL4 marks the wing blade region (red). Cell nuclei stained with DAPI (blue). e-h, Requirement of JAK/STAT signaling in compensatory proliferation. Wild-type (e) and stat92E⁻ (g) clones in adult eyes, marked by absence of red pigment. In eyes containing scrib⁻ clones confronted with wild-type cells (f), scrib⁻ cells (red) are mostly absent and size of the eye is largely normal. In eyes containing scrib⁻ clones (red) confronted with stat92E⁻ cells (h), the size of the eye is reduced. i, Model for the involvement of JNK and JAK/STAT signaling in intraclonal and interclonal cooperation between Ras^V12 and scrib⁻. See text for details.

The fact that both scrib⁻ clones and tissue damage induce overgrowth of Ras^V12 tissue suggests that compensatory proliferation in response to scrib⁻ cells could underlie cooperation in Ras^V12//scrib⁻ tumours. To test this, we studied the effect of confronting scrib⁻ cells with cells mutant for stat92E. When scrib⁻ clones are generated in eye-antennal discs, scrib⁻ cells in the adult eye are mostly absent ¹³ and the eye appears normal in size (Fig. 4e, f). When stat92E⁻ cells confront scrib⁻ cells, in contrast, the eye is greatly reduced (Fig. 4g, h; Supplemental Fig. 9), showing that stat92E⁻ cells cannot compensate for the loss of scrib⁻ cells. These data indicate a role for JAK/STAT signaling in tissue homeostasis through compensatory proliferation (see also Supplemental Figs. 9 and 10). Therefore, a mechanism to ensure recovery after damage explains the development of Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours and can mediate interclonal oncogenic cooperation (Fig. 4i).

We have used Drosophila to investigate how oncogenic cooperation between different cells can promote tumour growth and invasion. Our experiments, addressed to understanding interclonal cooperation in Ras^V12//scrib⁻ tumours, uncovered a two-tier mechanism by which scrib⁻ cells promote neoplastic development of Ras^V12 cells: (1) propagation of stress-induced JNK activity from scrib⁻ cells to Ras^V12 cells and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. Our findings, therefore, highlight the importance of cell interactions in oncogenic cooperation and tumour development. We also show that stress-induced JNK signaling and epigenetic factors such as tissue damage can contribute to tumour development in flies. Interestingly, tissue damage caused by conditions such as chronic inflammation has been linked to tumourigenesis in humans ^28,29. Furthermore, expression of the Unpaired cytokines promotes tumour growth (this study) as well as an antitumoural immune response ²², which parallels the situation in mice and humans ³⁰. Future research into phenomena such as compensatory growth and interclonal cooperation in Drosophila will provide valuable insights into the biology of cancer.